Antithrombin nucleotides and proteins from horn fly

ABSTRACT

Compositions and methods for preventing hematophagous infestation of cattle are provided, directed at isolated proteins with antithrombin activity and nucleotide sequences encoding the proteins. The protein named thrombostasin is isolated from the salivary glands of  Haematobia irritans . The compositions are useful as veterinary vaccines in prevention of blood-feeding in cattle by the infesting horn fly. The proteins of the invention are also useful in treatment of thrombosis.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. application Ser. No.09/376,113 filed Aug. 17, 1999, which claims the benefit of U.S. U.S.Provisional Application No. 60/097,227, filed Aug. 20, 1998, which ishereby incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

[0002] The invention relates to veterinary vaccines for prevention ofhematophagous infestation of cattle and medical treatment of thrombosis.

BACKGROUND OF THE INVENTION

[0003] Losses in livestock production in the United States due toectoparasite infestations have been estimated to exceed $2.26 billionannually (Byford et al. (1992) J. Anim. Sci. 70:597-602). Of the five tosix major arthropod pest species involved, the horn fly Haematobiairritans linnaeus is the most significant and widespread. Its annualeconomic impact on cattle production in the U.S.A. has been estimated at$730.3 million. In Canada, control of this ectoparasite in cattleproduction has been estimated to reduce losses by $71-107 million peryear using 1977 dollar values (Haufe and Weintraub (1985) Can. Entomol.117:901-907). Thus in North America, the annual economic impact oncattle production by this blood-sucking fly approaches $1 billion.

[0004] Physiological manifestations of hornfly infestation include anincrease in heart rates, respiration rates, and rectal temperatures.Additionally, water consumption and urine production are significantlyincreased as well as urinary nitrogen secretion. Blood cortisolconcentrations are also significantly increased. Decreased weight gain,increased activity, and decreased grazing have also been reported.(Schwinghammer et al. (1986) J. Econ. Entomol. 79:1010-1014).

[0005] The adult stage of both sexes of H. irritans are obligateectoparasites that blood-feed intermittently during the 24 hours of theday. Unlike other dipterous pests that are transient blood-feeders,(black flies, mosquitoes, horse flies, stable flies), the winged adultsof H. irritans remain on the bovine host and, when needing nourishment,recurrently insert their mouthparts into the skin to feed. Harris et al.(1974) Ann. Entomol. Soc. Am. 67:891-894, noted that under experimentalconditions, female horn flies spent an average of 163 minutes/dayfeeding; males averaged 96 minutes per day. Each female ingested anaverage of 17.1 mg of blood per day while males imbibed 12.1mg/individual due to the difference in feeding times (Harris and Frazer(1970) Ann. Entomol. Soc. Am. 63:1475-1476).

[0006] The scientific literature describing the salivary glandphysiology of H. irritans, particularly with reference to blood-feeding,is sparse. Hori et al. (1981) Appl. Ent. Zool, 16:16-23, has comparedseveral categories of digestive enzymes in the gut and salivary glandsof H. irritans with Stomoxys calcitrans (Linnaeus), the stable fly. Weakaminopeptidase activity was detected in H. irritans saliva, suggestingthat proteases and glycosidases in the gut are exclusively responsiblefor digestion of blood.

[0007] The horn fly Haematobia irritans linnaeus is a subspecies with H.i. exigua de Meijere, the buffalo fly that occurs in Australia andelsewhere in the southern hemisphere. Kerlin and Hughes (1992) Med. Vet.Entomol. 6:121-126, have compared enzymes in the saliva of fourparasitic arthropods—H. irritans exigua, Boophilus microplus(Canestrini), Aedes aegypti (Linnaeus), and Lucilia cuprina (Wiedemann)and noted differences in enzyme profiles of saliva between the fourspecies that apparently reflect their dissimilar feeding strategies.These differences were mainly in the type and levels of glycosidase andprotease activities. H. irritans exigua saliva, collected by serotoninstimulation and then evaluated by SDS polyacrylamide gelelectrophoresis, produced 7-8 bands by silver staining. Apyrase activityin saliva and salivary gland extracts (SGEs) of this species wasmarginally detectable, suggesting that this subspecies does not preventbovine platelet aggregation in the same way as many other blood-feedingarthropods (Ribeiro (1987) Ann. Rev. Entomol. 32:463-478).

[0008] Furthermore, investigation of immune response of cattle exposedto H. irritans exigua showed production of high levels of circulatingantibodies to some but not all of the buffalo fly antigens;nevertheless, flies feeding on previously exposed cattle did not exhibithigher mortality than those fed on unexposed cattle. (Kerlin andAllingham (1992) Vet. Parasitol. 43:115-129).

[0009] Elucidation of biochemical strategies adopted by blood-feedingarthropods has advanced in the past decade. Although the presence ofanticoagulants in saliva of hematophagous arthropods has been known forat least eight decades, only recently have some of the active componentsbeen purified and their molecular structures defined. It has becomeapparent that coagulation factors such as factors Xa and thrombin(factor II), which occur at a nexus in the coagulation cascade, arefrequently targeted.

[0010] Studies of saliva from several species of black flies havesuggested that specific enzyme targets may be associated with hostselection (Abebe et al. (1994)). For example, data for zoophagic speciesthat prefer cattle indicate that thrombin is an important targetmolecule whose inactivation may also prevent irreversible plateletaggregation in addition to impeding the coagulation cascade. See Hudson(1964) Can. J. Zool. 42:113-120, for Stomoxys calcitrans; and Parker andMant (1979) Thrombos. Haemostas (Stuttg.) 42:743-751, on G. morsitans(Westwood) saliva.

[0011] Because of the adverse impact of the above-describedectoparasitic infestation in cattle, there is a therapeutic and economicneed for preventing such infestation.

[0012] There is also need for treatment of thromboembolic diseases.Thromboembolic diseases are among the most important circulatorydiseases. A thrombus is a blood clot that partially or completely blocksblood flow through a blood vessel. An embolus is a thrombus that hasformed elsewhere in the body, broken free, and traveled to the sitewhere blockage occurs. Blockage in the brain results in a stroke, i.e.,a cerebral infarction, a localized area of dead cells. An embolus in alung can produce pulmonary embolism, one of the principal lung diseasesin bed-ridden patients. Bed ridden and elderly persons are alsoparticularly prone to thrombophlebitis, which is a blockage ofcirculation in a leg caused by an embolus. An embolus or thrombuslodging in one of the blood vessels serving the heart causes necrosis ofpart of the heart tissue, a myocardial infarction, commonly called aheart attack.

[0013] The initiating event of many myocardial infarctions is thehemorrhage into atherosclerotic plaques. Such hemorrhage often resultsin the formation of a thrombus (or blood clot) in the coronary arterywhich supplies the infarct zone. This thrombus is composed of acombination of fibrin and blood platelets. The formation of afibrin-platelet clot has serious clinical ramifications. The degree andduration of the occlusion caused by the fibrin-platelet clot determinesthe mass of the infarct zone and the extent of damage.

[0014] The formation of fibrin-platelet clots in other parts of thecirculatory system may be partially prevented through the use ofanticoagulants, such as heparin. Unfortunately, heparin has not beenfound to be universally effective in preventing reocclusion inmyocardial infarction victims in which the degree of blood vesselocclusion is greater than or equal to 70%, particularly in thosepatients with severe residual coronary stenosis. Among the morepromising of the agents are hirudin and its analogs, which bind to andinactivate thrombin. Hirudin has a theoretical advantage over heparin asan anti-thrombotic agent. Thrombin bound to thrombi or platelets isrelatively protected from inhibition by heparin while hirudin, at leastin vitro, is still effective. Other promising investigational agentsinclude fibrinogen receptor antagonists, which block plateletaggregation and dense granule release by a mechanism distinct from thatof aspirin, and inhibitors of thromboxane production.

[0015] There is therefore a need for additional antithrombin agentswhich exhibit low toxicity, little or no antigenicity, and a very shortclearance time from circulation.

SUMMARY OF THE INVENTION

[0016] Isolated proteins with antithrombin activity and nucleotidesequences encoding the proteins are provided. The protein namedthrombostasin is isolated from the salivary glands of Haematobiairritans, the blood-feeding horn fly. The provided proteins andnucleotides are particularly useful as veterinary vaccines in preventionof blood-feeding in cattle by the infesting horn fly.

[0017] The proteins of the invention are also useful in treatment ofthrombosis.

[0018] Methods of administering the proteins and nucleotide sequences ofthe invention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows molecular weight comparison of proteins incolony-versus field collected flies by relative mobility on SDS PAGE.

[0020]FIG. 2 depicts the recalcification time assay to test for anticoagulant activity in H. irritans saliva.

[0021]FIG. 3 shows the effect of H. irritans saliva on clotting ofFactor II deficient plasma.

[0022]FIG. 4 shows inhibition of thrombin hydrolysis of S238 by H.irritans saliva.

[0023]FIG. 5 shows HPLC purification of active salivary thrombostasin.

[0024]FIG. 6 shows SDS PAGE profile of HPLC purified salivaryanticlotting protein thrombostasin.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Methods and compositions for preventing hematophagy(blood-feeding) in cattle, and treatment of thrombosis in a mammal areprovided. The compositions comprise protein from the salivary gland ofthe hematophagous horn fly Haematobia irritans which, as described inYeates et al. (1999) Annu. Rev. Entemol. 44: 397-428, belong to thesuborder Cyclorrhapha of the order Diptera. Nucleotide sequencesencoding the antithrombin protein are additionally provided. The proteinhas been designated thrombostasin. The major function of the protein isto prevent coagulation by inhibiting the activity of thrombin (factorII).

[0026] By “hematophagy” is intended feeding on the blood of a hostorganism by another organism. By “hematophagous infestation” is intendeda host-parasite relationship comprising feeding on the blood of the hostby the parasite. By “thrombosis” is intended the formation, developmentor presence of a thrombus. By “antithrombin activity” is intended abiological activity that reduces or eliminates the procoagulant actionof thrombin; and/or inhibits thrombosis.

[0027] It is recognized that methods are available in the art to obtainthe complete coding sequence for the antithrombin protein of theinvention. Such methods are disclosed for example in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Plainview, N.Y.).

[0028] Substantially purified preparations of thrombostasin areprovided. Such substantially purified preparations include proteinssubstantially free of any compound normally associated with the proteinin its natural state. Such proteins can be assessed for purity bySDS-PAGE, chromatography, electrophoresis or other methods. See, M. P.Deutscher (ed.), Guide to Protein Purification, Academic Press, Inc.(1990).

[0029] The terms “substantially pure” or “substantially purified” arenot meant to exclude artificial or synthetic mixtures of the proteinwith other compounds. It is recognized that the antithrombin proteins ofthe present invention include those proteins homologous to, and havingessentially the same biological properties as, the antithrombin proteindescribed herein, and particularly the protein disclosed herein in SEQID NO: 2, SEQ ID NO:5, or SEQ ID NO:7. This definition is intended toencompass natural allelic variations in the genes. It is also recognizedthat “substantially purified” proteins of the present invention asdescribed herein can be of other species of origin, including but notlimited to other species of the suborder Cyclorrhapha.

[0030] The invention also provides fragments of the antithrombin proteinand nucleotide sequence disclosed in SEQ ID NOs: 1, 2, 4, 5, 6, and 7.Fragments of the protein may range in size from at least 10, 20, 30 ormore amino acids. Such fragments may retain biological activity orcomprise active regions of the protein.

[0031] Polynucleotide fragments may also range in size from at least 15,20, 30 or more contiguous nucleotides. The sequences find use ashybridization process or molecular markers.

[0032] Such fragments can be readily made by chemical methods includingcommercially available automated methods or by recombinant DNA methodsknown to the ordinarily skilled artisan, and described below. It isrecognized that biological functions of anti-hemostasis, including thoserelated to antithrombin anticoagulant activity and/or modulation ofimmune response may be carried out by the described fragments.

[0033] The invention additionally encompasses the nucleotide sequenceswhich encode the proteins of the invention. The nucleotide sequence ofthe PCR-cloned coding sequence from H. irritans is provided in SEQ IDNO: 1; however, it is recognized that cloned genes of the presentinvention can be of other species of origin, including but not limitedto other species of the suborder Cyclorrhapha.

[0034] DNAs which hybridize to the nucleotide sequence of theantithrombin gene from the horn fly are also an aspect of thisinvention. Conditions, which will permit other DNAs to hybridize to theDNA disclosed herein, can be determined in accordance with knowntechniques. For example, hybridization of such sequences may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at37° C.; conditions represented by a wash stringency of 40-45% Formamidewith 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42EC; and conditionsrepresented by a wash stringency of 50% Formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42EC, respectively, to DNA encoding thegenes disclosed herein in a standard hybridization assay. See J.Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2^(nd)ed.) (Cold Spring Harbor Laboratory).

[0035] In general, sequences which code for the antithrombin protein andhybridize to the nucleotide sequence disclosed herein will be at least40% homologous, about 60% to 70% homologous, and even about 80%, 85%,90% homologous or more with the disclosed sequences. Such sequences aresubstantially homologous to the nucleotide sequences disclosed hereinand encompassed by the invention. Further, the amino acid sequences ofthe antithrombin proteins isolated by hybridization to the DNA'sdisclosed herein are also an aspect of this invention. The degeneracy ofthe genetic code, which allows different nucleic acid sequences to codefor the same protein or peptide, is well known in the literature. See,e.g., U.S. Pat. No. 4,757,006.

[0036] The hybridization probes may be cDNA fragments oroligonucleotides, and may be labeled with a detectable group as known inthe art. Pairs of probes which will serve as PCR primers for theantithrombin gene or a protein thereof may be used in accordance withthe process described in U.S. Pat. Nos. 4,683,202 and 4,683,195.

[0037] The polypeptides of the invention may be subject to one or morepost-translational modifications such as sulphation, COOH-amidation,acylation or chemical alteration of the polypeptide chain.

[0038] It is recognized that the nucleotide and peptide sequences of theinvention may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the peptides and proteins can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel, T.(1985) Proc. Natl. Acad. Sci. U.S.A 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra (eds.) Techniques in Molecular Biology, MacMillan PublishingCompany, NY (1983) and the references cited therein. Thus, thenucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant. Likewise, the peptides andproteins of the invention encompass both naturally occurring andmodified forms thereof. Such variants will continue to possess thedesired activity. It is recognized that the mutations that will be madein the DNA encoding the variant must not place the sequence out ofreading frame and preferably will not create sequences deleterious toexpression of the gene product. See, EP Patent Application, PublicationNo. 75,444.

[0039] The proteins of the invention include the naturally occurringforms as well as variants thereof. These variants will be substantiallyhomologous and functionally equivalent to the native protein. As usedherein, two proteins (or a region of the proteins) are “substantiallyhomologous” when the amino acid sequences are typically at least about40%, more typically at least about 60%-70%, and most typically at leastabout 80%, 85%, 90% or more identical. A substantially homologous aminoacid sequence, according to the present invention, will be encoded by anucleic acid sequence hybridizing to the nucleic acid sequence, orportion thereof, of the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:6, or otherwise described herein under stringentconditions as more fully described below.

[0040] Thus, a variant of a native protein is “substantially homologous”to the native protein when at least about 40%, more preferably at leastabout 60%-70%, and most preferably at least about 80%, 85%, 90%, or moreof its amino acid sequence is identical to the amino acid sequence ofthe native protein. A variant may differ by as few as 1, 2, 3, or 4amino acids. A variant polypeptide can differ in amino acid sequence byone or more substitutions, deletions, insertions, inversions, fusions,and truncations or a combination of any of these.

[0041] By “functionally equivalent” is intended that the sequence of thevariant defines a chain that produces a protein having substantially thesame biological activity as the native protein of interest. Suchfunctionally equivalent variants that comprise substantial sequencevariations are also encompassed by the invention. Thus a functionallyequivalent variant of the native protein will have a sufficientbiological activity to be therapeutically useful. By therapeuticallyuseful is intended effective in achieving a therapeutic goal asdiscussed below.

[0042] Methods are available in the art for determining functionalequivalence. Biological activity can be measured using assaysspecifically designed for measuring activity of the native protein,including assays described in the present invention. Additionally,antibodies raised against the biologically active native protein can betested for their ability to bind to the functionally equivalent variant,where effective binding is indicative of a protein having conformationsimilar to that of the native protein.

[0043] Variant polypeptides can be fully functional or can lack functionin one or more activities. Thus, in the present case, variations canaffect the function, for example, of one or more of the modules,domains, or functional subregions of the proteins and polypeptides ofthe invention.

[0044] Fully functional variants typically contain only conservativevariation or variation in non-critical residues or in non-criticalregions. Functional variants can also contain substitution of similaramino acids, which result in no change or an insignificant change infunction. Alternatively, such substitutions may positively or negativelyaffect function to some degree.

[0045] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region. As indicated,variants can be naturally-occurring or can be made by recombinant meansor chemical synthesis to provide useful and novel characteristics forthe polypeptide.

[0046] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)). The latter procedure introduces single alanine mutations atevery residue in the molecule. The resulting mutant molecules are thentested for biological activity. Sites that are critical can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0047] The invention further encompasses variant polynucleotides, andfragments thereof, that differ from the nucleotide sequence shown in SEQID NO:1, SEQ ID NO:4, or SEQ ID NO:6, or otherwise described herein, dueto degeneracy of the genetic code and thus encode the same protein asthat encoded by the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:4, or SEQ ID NO:6 or otherwise described herein.

[0048] The invention also provides nucleic acid molecules encoding thevariant polypeptides described herein. Such polynucleotides may benaturally occurring, such as allelic variants (same locus), homologs(different locus), and orthologs (different organism), or may beconstructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to polynucleotides, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions.

[0049] Variation can occur in either or both the coding and non-codingregions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0050] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. These variants comprise a nucleotidesequence encoding a protein that is at least typically about 40%, moretypically at least about 60%-70%, and most typically at least about 80%,85%, 90% or more homologous to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO: 6 or otherwise described herein, or afragment of this sequence. Such nucleic acid molecules can readily beidentified as being able to hybridize under stringent conditions, to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO: 6 orotherwise described herein, or a fragment of the sequence. It isunderstood that stringent hybridization does not indicate substantialhomology where it is due to general homology, such as poly A sequences,or sequences common to all or most proteins in an organism or class ofproteins.

[0051] To determine the percent homology of two amino acid sequences, orof two nucleotide sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of oneprotein or nucleic acid for optimal alignment with the other protein ornucleic acid). The amino acid residues or nucleotides at correspondingamino acid positions or nucleotide positions are then compared. When aposition in one sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the other sequence, then themolecules are homologous at that position. As used herein, amino acid ornucleic acid “homology” is equivalent to amino acid or nucleic acid“identity”. The percent homology between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent homology equals the number of identical positions/total numberof positions times 100).

[0052] The invention also encompasses proteins or polypeptides having alower degree of identity but having sufficient similarity so as toperform one or more of the same functions performed by the antithrombinproteins described herein. Similarity is determined by conserved aminoacid substitution. Such substitutions are those that substitute thegiven amino acid in a polypeptide by another amino acid of likecharacteristics. Conservative substitutions are likely to bephenotypically silent. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247:1306-1310 (1990).

[0053] Both identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).Preferred computer program methods to determine identify and similaritybetween two sequences include, but are not limited to, GCG programpackage (Devereux, J. (1984) Nuc. Acids Res. 12(1):387), BLASTP, BLASTN,and FASTA (Atschul, S. F. (1990) J. Molec. Biol. 215:403); utilizing thedefault parameters available within the programs. By substantialsequence similarity, identity or homology is intended sequences havingat least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more similarity.

[0054] DNA sequences can also be synthesized chemically or modified bysite-directed mutagenesis to reflect the codon preference of the hostcell and increase the expression efficiency.

[0055] The proteins of the invention can be engineered in accordancewith the present invention by chemical methods or molecular biologytechniques. Molecular biology methods are most convenient since proteinscan be engineered by manipulating the DNA sequences encoding them.Genomic DNA, cDNA, synthetic DNA, and any combination thereof may beused for this purpose. Genomic DNA sequences or cDNA sequences encodingproteins can be isolated based on the amino acid sequence of proteins orcertain protein properties. Many methods of sequence isolation are knownin the art of molecular biology. See particularly Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Plainview, N.Y.), herein incorporated by reference.

[0056] To produce an antithrombin polypeptide by recombinant DNAtechnology, a gene encoding a polypeptide of the invention is prepared.The DNA coding sequence typically does not contain introns. The DNAsequence is isolated and purified, the gene is inserted in an expressionvector able to drive expression and production of the recombinantproduct. The DNA sequence may be a cDNA sequence, or alternatively asynthetic DNA sequence. A synthetic gene is typically prepared bychemically synthesizing oligonucleotides that, in total, correspond tothe desired gene. The synthesized oligonucleotides are then assembled toobtain the gene.

[0057] If desired, the gene sequence may be modified by site-directedmutagenesis to introduce one or more coding changes. Typically, a geneis constructed with restriction sites at each end to facilitate itssubsequent manipulation.

[0058] The DNA sequence may be constructed to comprise a leader peptide.The leader peptide is capable of directing secretion of the polypeptidefrom cells in which the polypeptide is to be expressed. The sequenceencoding the leader peptide is typically fused to the 5′-end of the DNAsequence encoding the polypeptide. Leader sequences are known in the artand include the OmpA leader peptide, the leader peptide of vesicularstomatitis virus G protein (VSV G protein). The OmpA leader is usefulwhen expression is in a bacterial host, such as E. coli while the VSVGprotein is useful when expression is in insect cells.

[0059] The DNA sequence may be constructed to comprise a cleavable siteto release the polypeptide of the invention. A DNA sequence may be usedwhich encodes a carrier polypeptide sequence fused via a cleavablelinkage to the N-terminus of a polypeptide of the invention. Thecleavable linkage may be one cleavable by cyanogen bromide.

[0060] For expression of the polypeptides, an expression vector isconstructed which comprises a DNA sequence encoding the polypeptidewhich is capable of expressing the polypeptide in a suitable host.Appropriate transcriptional and translational control elements areprovided, including a promoter for the DNA sequence, a transcriptionaltermination site, and translation start and stop codons. The DNAsequence is provided in the correct frame such as to enable expressionof the polypeptide to occur in a host compatible with the vector.

[0061] The expression vector typically comprises an origin ofreplication and, if desired, a selectable marker gene such as antibioticresistance. The expression vector may be a plasmid, a virus,particularly a baculovirus, and the like.

[0062] Once the nucleotide sequences encoding the antithrombin proteinsof the invention have been isolated, they can be manipulated and used toexpress the protein in a variety of hosts including other organisms,including microorganisms.

[0063] Once the nucleotide sequence is identified and known, thoseskilled in the art can produce large quantities of the protein fortherapeutic use. Accordingly, recombinant protein and methods forproducing the recombinant protein are encompassed by the presentinvention. In this manner, the nucleotide sequence encoding theantithrombin protein can be utilized in vectors for expression invarious types of host cells, including both procaryotes and eucaryotes,to produce large quantities of the protein, or active analogues, orfragments thereof, and other constructs having antithrombin activity.

[0064] Generally, methods for the expression of recombinant DNA areknown in the art. See, for example, Sambrook et al. (1989) MolecularCloning, Cold Spring Harbor Laboratory. Additionally, host cells andexpression vectors, such as the baculovirus expression described in U.S.Pat. No. 4,745,051 and U.S. Pat. No. 4,879,236. In general, abaculovirus expression vector comprises a baculovirus genome containingthe gene to be expressed inserted into the polyhedron gene at a positionranging from the polyhedron transcriptional start signal to the ATGstart site and under the transcriptional control of a baculoviruspolyhedron promoter.

[0065] A broad variety of suitable procaryotic and microbial vectors areavailable. Likewise, the promoters and other regulatory agents used inexpression of foreign proteins are available in the art. Promoterscommonly used in recombinant microbial expression vectors are known inthe art and include the beta-lictamase (penicillinase) and lactosepromoter systems (Chang et al. (1978) Nature 275:615 and Goeddel et al.(1979) Nature 281:544); A tryptophan (TRP) promoter system (Goeddel etal. (1980) Nucleic Acids Res. 8:4057 and the EPO Application PublicationNo. 36,776); and the Tac promoter (DeBoer et al. (1983) Proc. Natl.Acad. Sci. U.S.A, 80:21). While these are commonly used, other microbialpromoters are available. Details concerning nucleotide sequences of manyhave been published, enabling a skilled worker to operably ligate themto DNA encoding the protein in plasmid or viral vectors. See, forexample, Siedenlist et al. (1980) Cell 20:269.

[0066] Eucaryotic host cells such as yeast may be transformed withsuitable protein-encoding vectors. See, e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al. (1979) Nature 282:9;Kingsman et al. (1979) Gene 7:141; Tschemper et al. (1980) Gene 10:157).This plasmid contains the trp1 gene, which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics 85:12). Thepresence of the trp1 lesion in the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

[0067] Suitable promoter sequences for use in yeast vectors include thepromoters for metallothionein, alcohol dehydrogenase, adenylate cyclase,3-phosphoglycerate kinase (Hitzeman et al. (1980) J. Biol. Chem.255:2073) and other glycolytic enzymes (Hess et al. (1968) J. Adv.Enzyme Reg. 7:149; and Holland et al. (1978) Biochemistry 17:4900) suchas enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al. EPO Publn. No. 73,657.

[0068] The invention provides antibody preparations that selectivelybind the proteins of the invention, or any variants or fragments thereofas described. An antibody is considered to selectively bind, even if italso binds to other proteins that are not substantially homologous withthe antithrombin protein. These other proteins share homology with afragment or domain of the antithrombin protein giving rise to antibodiesthat bind to both proteins by virtue of the homologous sequence. In thisaspect, it is recognized that antibody binding to the antithrombinprotein is still selective.

[0069] The preparations encompass monoclonal or polyclonal antibodies,intact antibodies or fragments thereof (e.g. Fab), purified preparationssuch as affinity-purified preparations, or less pure preparations suchas ascites fluid, sera and the like. Methods for raising antibodies arewell known in the art and include but are not limited to those describedin Harlow and Lane ((1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press), the contents of which are herein incorporatedby reference. The invention also embodies antibody preparations whichneutralize biological functions of the provided proteins, variants orfragments thereof. Such functions include but are not limited toantithrombin activity. The invention also provides compositions capableof modulating the immune response. By modulating the immune response isintended a determinable change in the immune system of a host organismeffected by administering the herein described compositions of theinvention to that host. Working examples of such modulation of immuneresponse, as well as methods of making and assessing selectivity ofantibody preparations are provided in the Experimental section of thisapplication, and are herein incorporated by reference.

[0070] The compositions of the present invention find therapeutic use asveterinary vaccines in treatment of hematophagy in a mammal. The methodscomprise administering to the mammal a veterinary vaccine comprising atherapeutically effective amount of the compositions of the invention.In this aspect, a therapeutically effective amount is intended as thatamount which effects a determinable reduction, amelioration, eliminationor prevention of hematophagous infestation in the mammal to which thevaccine of the present invention was administered. While the vaccines ofthe invention can be used with any mammal, of particular interest arelivestock, more particularly, horses, cattle, and the like. Thecompositions are useful for vaccination against the hematophagous fly ofthe suborder Cyclorrhapha, more particularly of the species Haematobiairritans, even more particularly of the subspecies irritans or exigua.However, the invention vaccination against any hematophagous organismwhere such vaccination using compositions and methods of the presentinvention is therapeutically effective.

[0071] For veterinary applications, the compositions of the inventioncan be formulated into any acceptable pharmaceutical preparation asdescribed below or any other acceptable preparation for veterinary use.In one embodiment of the invention, the vaccines comprisetherapeutically effective amounts of the proteins of the invention, orany variant or fragment thereof as described herein.

[0072] In a preferred embodiment, the vaccines comprise the nucleotidecompositions of the invention as described herein. As described by Coxet al. (1993) J. Virol. 67:5664-5667; Fynan et al. (1993) Proc. Natl.Acad. Sci. USA 90:11478-11482; and Lewis et al. (1997) Vaccine15:861-864; and reviewed by Robinson (1997) Vaccine 15:785-787; andTighe et al. (1998) Immunol. Today 19:89-97, the contents of all ofwhich are herein incorporated by reference, nucleic acid vaccines can bereadily constructed and produced. In general, target DNA sequencesencoding the protein to be used as an immunogen are cloned intoeukaryotic expression vectors. The constructed plasmid is grown inbacteria and purified. The purified plasmid DNA is then directlyinjected into the animal generally by intramuscular injection, but alsoby intradermal injection; where its expression by cells in theinoculated host produces the target protein, thereby raising an immuneresponse. See, for example, Cox et al. (1993) J. Virol. 67:5664-5667,herein incorporated by reference. Nanogram levels of DNA-expressedprotein may be utilized to stimulate an immune response and protectagainst infectious agents achieved by skin, muscle and intravenousinoculations of DNA. See, for example, Fynan et al. (1993) Proc. Natl.Acad. Sci. USA 90:11478-11482; Cox et al. (1993) J. Virol. 67:5664-5667,herein incorporated by reference. Such plasmids introduced byintramuscular or intradermal injection stimulate a protective responsethat abrogates clinical disease following challenge.

[0073] The compositions of the present invention can be formulated intopharmaceutical preparations for therapeutic use as antithrombin agents.Such compositions find use in the treatment of venous thrombosis,vascular shunt occlusion and thrombin-included disseminatedintravascular coagulation.

[0074] The compositions of the invention can be used alone or incombination with other antithrombin and therapeutic agents includingveterinary agents such as vaccines. Other agents are known in the art.

[0075] The antithrombin compositions can be formulated according toknown methods to prepare pharmaceutically useful compositions, such asby admixture with a pharmaceutically acceptable carrier vehicle.Suitable vehicles and their formulation are described, for example, inRemington's Pharmaceutical Sciences 19th ed., Osol, A. (ed.), MackEaston Pa. (1980). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the antithrombin protein, eitheralone, or with a suitable amount of carrier vehicle.

[0076] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the compositions. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carbosymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules.

[0077] Another possible method for controlling the duration of actioncomprises incorporating the therapeutic agents into particles of apolymeric substance such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, itis possible to entrap the therapeutic agents in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, by the use of hydroxymethyl cellulose orgelatin-microcapsules or poly(methylmethacrylate) microcapsules,respectively, or in a colloid drug delivery system, for example,liposomes, albumin, microspheres, microemulsions, nanoparticles,nanocapsules, or in macroemulsions. Such teachings are disclosed inRemington's Pharmaceutical Sciences (1980).

[0078] In more specific embodiments, a polypeptide of the invention maybe converted into a pharmaceutically acceptable salt. It may beconverted into an acid additional salt with an organic or inorganicacid. Suitable acids include acetic, succinic and hydrochloric acid.Alternatively, the peptide may be converted into a carboxylic acid saltsuch as the ammonium salt or an alkali metal salt such as the sodium orpotassium salt.

[0079] A polypeptide or pharmaceutically acceptable salt thereof may beused in a pharmaceutical composition, together with a pharmaceuticallyacceptable carrier or excipient therefore. Such a formulation istypically for intravenous administration (in which case the carrier isgenerally sterile saline or water of acceptable purity). A polypeptidecan therefore be used for the therapy and prophylaxis of thrombosis andthromboembolisms in a human or other mammal, including the prophylaxisof post-operative thrombosis, for acute shock therapy (for example forseptic or polytraumatic shock), for the therapy of consumptioncoagulopathics, in hemodialyses, haemoseparations and in extracorporealblood circulation. In one embodiment of the invention, the polypeptideor salt thereof can be coadministered with a plasminogen activator, suchas tissue plasminogen activator.

[0080] The dosage depends especially on the specific form ofadministration and on the purpose of the therapy or prophylaxis. Thesize of the individual doses and the administration regime can best bedetermined by way of an individual judgment of the particular case ofillness; the methods of determining relevant blood factors required forthis purpose are familiar to the person skilled in the art. Normally, inthe case of an injection the therapeutically effective amount of thecompounds according to the invention is in a dosage range of fromapproximately from 0.005 or 0.01 to approximately 0.05 or 0.1 mg/kg bodyweight, preferably from approximately 0.01 to approximately 0.05 mg/kgbody weight.

[0081] The administration is effected by intravenous, intramuscular orsubcutaneous injection. Accordingly, pharmaceutical compositions forparenteral administration in single dose form contain per dose,depending on the mode of administration, from approximately 0.4 toapproximately 7.5 mg of the compound according to the invention. Inaddition to the active ingredient these pharmaceutical compositionsusually also contain a buffer, for example a phosphate buffer, which isintended to keep the pH value between approximately 3.5 and 7, and alsosodium chloride, mannitol or sorbitol for adjusting the isotonicity. Thepreparations may be freeze-dried or dissolved. An antibacterially activepreservative may be included, for example from 0.2 to 0.3%4-hydroxybenzoic acid methyl ester or ethyl ester.

[0082] A composition for topical application can be in the form of anaqueous solution, lotion or gel, an oily solution or suspension or afat-containing or, especially, emulsified ointment. A composition in theform of an aqueous solution is obtained, for example, by dissolving theactive ingredients according to the invention, or a therapeuticallyacceptable salt thereof, in an aqueous buffer solution of from e.g., pH4 to pH 6.5 and, if desired, adding a further active ingredient, forexample an anti-inflammatory agent, and/or a polymeric binder, forexample polyvinylpyrrolidone, and/or a preservative. The concentrationof active ingredients is from approximately 0.1 to approximately 1.5 mg,preferably from 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.

[0083] An oily form of administration for topical application isobtained, for example, by suspending the active ingredient according tothe invention, or a therapeutically acceptable salt thereof, in an oil,optionally with the addition of swelling agents, such as aluminumstearate, and/or surfactants (tensides) having an HLB value(“hydrophilic-lipophilic balance”) of below 10, such as fatty acidmonomers of polyhydric alcohols, for example glycerin monostearate,sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. Afat-containing ointment is obtained, for example, by suspending theactive ingredient according to the invention, or a salt thereof, in aspreadable fatty base, optionally with the addition of a tenside havingan HLB value of below 10. An emulsified ointment is obtained bytriturating an aqueous solution of the active ingredient according tothe invention, or a salt thereof, in a soft, spreadable fatty base withthe addition of a tenside having an HLB value of below 10. All theseforms for topical application can also contain preservatives. Theconcentration of active ingredient is from approximately 0.1 toapproximately 1.5 mg, preferably from 0.25 to 1.0 mg, in approximately10 g of base.

[0084] In addition to the compositions described above andpharmaceutical compositions analogous thereto that are intended fordirect medicinal use in the body of a human or a mammal, the presentinvention relates also to pharmaceutical compositions and preparationsfor medicinal use outside the living body of humans or mammals. Suchcompositions and preparations are used especially as anticoagulantadditives to blood that is being subjected to circulation or treatmentoutside the body (for example haemoseparation). Such preparations, suchas stock solutions or alternatively preparations in single dose form,are similar in composition to the injection preparations describedabove; however, the amount of concentration of active ingredient isadvantageously based on the volume of blood to be treated or, moreprecisely, on its thrombin content. Depending on the specific purpose,the suitable dose is from approximately 0.01 to approximately 1.0 mg ofthe active ingredient/liter of blood, although the upper limit may stillbe exceeded without risk as the agent is harmless even in relativelyhigh amounts.

Experimental

[0085] Collection and Rearing of H. irritans

[0086] Pupae were shipped from the U.S.D.A. Livestock Insects ResearchLaboratory in Kerrville, Tex., on a biweekly basis and stored at 4ECuntil needed. They were removed and placed in stainless steel cages(18″×18″×18″) at room temperature (21-22EC) with 16:8 hours (L:D) topromote emergence of adults. An absorbent cotton pad was placed on topof each cage and used as a wick to supply fresh blood to adults on adaily basis.

[0087] Wild-caught adults collected from the University of Arizona dairyherd and from the Auburn University beef and dairy herds were used forsome assays. They were transported to the laboratory within an hour ofcollection and maintained as above prior to experimentation.

[0088] Recovery of Salivary Glands

[0089] Both sexes of H. irritans are obligate blood feeders and theirsalivary glands are similar in morphology and location in the body tostable flies (Stomoxys calcitrans) and tsetse flies (Glossina spp.) Thefollowing protocol was used for dissection of glands: (a) the fly was“knocked down” with humidified CO2, passed briefly through a 70% ethanol(ETOH) bath, and then rinsed in deionized water; (b) it was placed on aclean glass slide in a drop of chilled 0.15M saline and the legs, wingsand head were removed. The thorax was split sagittally using a razorblade or scalpel; (c) the fly was then transferred to a fresh drop ofchilled saline in a watch glass or a small dish filled with paraffin.Using minute dissecting needles, the two halves of the thorax were thenpeeled back; (d) using forceps, the abdominal cuticle was pulled away,exposing the internal organs. The salivary glands were then teased awayfrom the gut tissue. The anterior end of the gut (the cardia) wasclipped and then gut-salivary gland assembly withdrawn by pulling itthrough the abdomen-thorax constriction; (e) the glands were then teasedaway from the gut, rinsed once in cold saline and transferred to anEppendorf to be kept in ice for collection, and then frozen at −70EC.

[0090] Preparation of Salivary Gland Extracts

[0091] Salivary gland extracts (SGEs) were prepared as described by Cuppet al. (1993) J. Insect Physiol. 39:817-821, or by sonication. For theformer method, glands were homogenized in a 1:1 mixture of 0.15 M NaClsolution and 0.1% Triton X-100 was added to the thawed sample, which wasthen refrozen. Extracts were prepared by thawing the solubilized sample,vortexing it for 30 seconds and then centrifuging it at 14,000×g for 30seconds at 4° C. For the latter method, sonic disruption of glands wasobtained using 70% cycle and 70% power output of a Sonifier 450 (BransonUltrasonics, Danbury, Conn.) for 2 minutes. Eppendorf tubes with glandswere thawed and the contents disrupted by holding the tip of each tubeto the base of the sonic probe immersed in an ice bath to disperse heat.Salivary gland extracts were transferred to a new tube following removalof cell fragments by centrifugation at ≈12,000×g for 5 minutes at 4EC.The amount of protein per individual gland was determined using a BCAprotein assay kit (Pierce, Rockford, Ill.). Initial measurement ofsoluble protein obtained from sonicated H. irritans salivary glands was0.54±0.09 μg/pair of glands for females and 0.63±0.02 μg/pair of glandsfor males.

[0092] Collection of Saliva

[0093] To determine antihemostatic activity attributable specifically tosalivary secretion, two methods were joined which have been usedpreviously for the buffalo fly (Kerlin and Hughes (1992) Med. Vet.Entomol. 6:121-126) and mosquitoes (Hurlbut (1966) Am. J. Trop. Med.Hyg. 15:989-993) to collect saliva from these insects. Adult flies, heldat room temperature, were starved for 24 hours to insure that secretionswere retained in the salivary glands and that all gut contents weredigested. The latter precaution is necessary since muscoid flies oftenregurgitate during feeding. The flies were then anesthetized withhumidified CO2 and their wings removed with microdissecting scissors.The dealated flies were then glued to applicator sticks so that theirmouth parts could be positioned into a capillary tube containing mineraloil. Just prior to this step, each fly was injected with 1 μl of 80 mMserotonin. The fly's proboscis was then inserted into the oil which,because of its difference in viscosity with saliva, served as acollecting medium for the serotonin-induced secretions. Salivationusually began within 30-60 seconds and the saliva could be easily seenas a clear aqueous droplet when it was expelled into the oil.

[0094] Gel Electrophoresis

[0095] Unless otherwise indicated, proteins were resolved on 15%polyacrylamide/SDS gels (SDS PAGE) by the method of Laemmli (1970)Nature 227:680-685, and visualized by silver staining (Bassam et al.(1991) Annal. Biochem. 196:80-83). Stained gels are scanned fordensitometry analysis of band migration and staining intensity (PersonalDensitometer S.I., ImageQuaNT for Windows NT, Molecular Dynamics,Sunnyvale, Calif.).

[0096] Proteins in Saliva

[0097]FIG. 1 depicts molecular weight comparison of proteins in salivaof colony (lane C) versus field-collected (Lane B) flies by relativemobility on SDS PAGE. Molecular weight standards in the 10-220 kDa rangeare shown in lanes A and D. A very similar profile is observed exceptfor the presence of a light band at ≈36 KDa in field-collected flies.However, the concentration of proteins in the saliva of the 30field-collected flies (B), as determined by relative intensity ofstaining of bands, exceeds that of corresponding bands in the saliva of84 colony flies (C). This difference was observed routinely onsilver-strained gels and indicates that field populations of H. irritansproduce greater concentrations of salivary proteins than do flies fromthis colonized strain.

[0098] Apyrase Activity

[0099] Apyrase activity in SGEs was tested using a standard assay (seeCupp et al. (1993) J. Insect Physiol. 39:817-821). This enzyme rapidlydegrades adenosine triphosphate (ATP) and adenosine diphosphate (ADP) tothe monophosphate, thereby eliminating a crucial chemical signal thatordinarily promotes platelet aggregation. Extracts were prepared fromwild caught male and female flies which were maintained on water for 48hrs prior to dissection. Activity in this enzyme in SGEs was marginallydetectable in H. irritans (2.59±0.21 milliUnits/pair of salivary glandequivalents). This lack of apyrase activity was also confirmed by theinability of H. irritans saliva to affect ADP-induced aggregation ofplatelets in bovine platelet-rich plasma (unpublished observations).Thus, apyrase activity was eliminated as a mechanism of hematophagy byH. irritans.

[0100] Erythema Activity

[0101] We evaluated the potential of H. irritans saliva to induceerythema, using intradermal injections of SGEs or by direct feeding ofmale and female flies on the shaved back of a New Zealand White rabbit.As a control, we also injected Simulium vittatum SGEs which produce apersistent erythema within 15 min of intradermal delivery (Cupp et al.(1994) Am. J. Trop. Med. Hyg. 50:235-240). A colonized strain of S.vittatum served as a source of salivary gland material (Bernardo et al.(1986) Ann. Entomol. Soc. Am. 79:610-621). No erythema was produced byeither male or female H. irritans saliva, whether injected as an SGE ordelivered by bite. Simulium vittatum SGE produced a visible erythemawithin 15 minutes. Thus, erythma activity was eliminated as a mechanismof hematophagy by H. irritans.

[0102] Other Vasodilative Activity

[0103] Studies were conducted to detect the presence of vasodilativeactivity in H. irritans SGEs or saliva using tension measurements of ratstomach (assay for prostaglandin) and rabbit aortic strips, with andwithout intact endothelium (see Ribeiro et al. (1992) Exp. Parasitol.74:112-116; Ribeiro et al. (1994) J. Med. Entomol. 31:747-753). Todetect bradykinin or histamine activity in H. irritans SGEs, the assayfollowed the procedure of Webster and Prado (1970) which uses thecontraction in vitro of guinea pig ileum as a direct bioassay of kininactivity. Normal responses to test substances (prostaglandin E2 for ratstomach strips and norepinephrine or acetyl choline for rabbit aorticstrips) were obtained, while H. irritans SGE showed no vaso-activity.Initially, collections of induced saliva did show activity in the ratstomach strip assay but this was lost when methysergide maleate wasincluded (Pertz and Eich (1992) Navnyn Schmiedebergs Arch. Pharmacol.345:394-401. This substance is a known inhibitor of serotonin, thecompound used to elicit salivation by the fly. The presence of activityin serotonin-induced saliva, but not in SGE, indicated that theserotonin activity in those samples was derived from the injectedcompound used to elicit salivation. Extraction of H. irritans SGE toenhance detection of prostaglandin activity confirmed the negativeresults of the earlier vasodilatory study. No salivary activity wasdetected in the guinea pig ileum assay for bradykinin or histamine.Thus, the tested vasodilative activities were eliminated as mechanismsof hematophagy by H. irritans. The inability of hornfly SGE to elicitvasodilation when injected intradermally into the shaved skin of NZWrabbits, in vivo, was confirmed using laser doppler perfusion imaging.

[0104] Anti-coagulant Activity

[0105] The re-calcification time assay was chosen to screen foranticoagulant activity, as this general assay can detect inhibitors thatattack at any of the three major arms of the coagulation cascade, theextrinsic pathway, the intrinsic pathway and the final common pathway.Salivary gland extracts were prepared from both male and female H.irritans and from female S. vittatum. SGEs from the latter species wereused as a positive control because the same re-calcification time assayhad been used previously to detect anticoagulant activity in thatspecies (Abebe et al. (1994) J. Med. Entomol. 31:908-911). Salivarygland extracts of female H. irritans were as potent as those of S.vittatum in delaying the re-calcification time of standard plasma asshown in FIG. 2. Male SGEs also delayed re-calcification time (data notshown). Comparable inhibition occurred in spite of the fact thatmeasured protein contents were 50% lower in extracts of H. irritans.Thus, this anti-coagulant activity was the only anti-hemostatic activitydetected for the horn fly, H. irritans.

[0106] Anti-hemostatic Specificity

[0107] The recalcification time assay can detect inhibition of any stepin the cascade of reactions that ultimately lead to blood-clotting(coagulation), and thus it is a useful general test to screen for thepresence of an unknown inhibitor. Because blood-clotting is the resultof a series of reactions, horn fly saliva could delay clotting byinhibiting a specific step in the blood-clotting cascade or,alternatively, delay the normal rate of hemostasis by dissolving a clotafter it was formed (fibrinolytic activity).

[0108] For analytical purposes the clotting reactions are typicallygrouped into three sub-pathways which are monitored by differentclotting assays; i.e., the intrinsic (activated partial thromboplastintime test=APTT), the extrinsic (prothrombin time test=PTT) and the finalcommon pathway (thrombin time=TT). Recalcification time, PTT, TT andAPTT assays are well known by those ordinarily skilled in the art. Forexample, see Biggs et al. ((1962) Human Blood Coagulation And ItsDisorders, 3rd ed., Blackwell Scientific Publications, Oxford) forrecalcification time assays, and Turgeon M. L. ((1993) ClinicalHematology. Theory and Procedures, 2nd ed., Little, Brown and Company,Boston) for APTT, PTT and TT assays. APTT II is a modification of theAPTT I test and is more sensitive.

[0109] Using these tests, several properties of horn fly anticlottingactivity were determined as shown in Table 1:1) Horn fly salivary glandextracts or saliva caused delay in clotting of all the tests, indicatingthat at least one inhibitor is present that works in the final commonpathway, i.e. after the formation of thrombin from prothrombin. 2)Saliva from wild-type flies contains more inhibitor activity than salivacollected from the same number of colony flies. 3) Inhibitor activity incolony flies held for 48 hours after emergence is greater than at 24hours post-emergence. TABLE 1 Delay in blood clotting by Haematobiairritans salivary gland extracts (SGE) or serotonin-induced saliva.Source # flies Type of Assay % of Control* SGE-colony 1 Recalcification106 SGE-colony 2 Recalcification 128 Saliva-colony (24 h) 4Recalcification 143 Saliva-colony (48 h) 4 Recalcification 175Saliva-wild type 1 Recalcification 127 Saliva-wild type 2Recalcification 161 SGE-colony 1 APTT-I 113 SGE-colony 2 APTT-I 149Saliva-colony 1 APTT-I 112 SGE-colony 1 APTT-II 144 Saliva-wild type 1APTT-II 210 SGE-colony 1 PTT 120 SGE-colony 2 PTT 140 Saliva-wild type 1PTT 156 SGE-colony 1 TT ND SGE-colony 2 TT 109 Saliva-wild type 1 TT 158

[0110] Inhibition of clotting in the TT assay by horn fly salivaindicates that a reaction occurring after the formation of thrombin istargeted. Two reactions occur after that point 1) the formation offibrin monomers by the action of thrombin (factor II) on fibrinogen and2) the cross-linking of fibrin monomers by the action of factor XIII.Thrombin is also involved in the activation of factor XIII. Thus,thrombin (factor II) was a probable target of horn fly saliva. To testthis possibility, clotting times of plasma that had been depleted offactor II by using specific antibodies (Sigma Chemical, St. Louis, Mo.)were determined. Addition of increasing amounts of normal plasma,(containing factor II), decreased the time for clotting as measured bythe PTT assay (FIG. 3, −saliva). When horn fly saliva (equivalent to 2flies) was added with the increasing amounts of normal plasma (FIG. 3,+saliva), the percentage delay in clotting time increased withincreasing amounts of factor II (present in normal plasma). This patternindicated that saliva contained a specific inhibitor of factor II.

[0111] Thrombin clotting action can be measured using a syntheticsubstrate (S238, American Diagnostica Inc., Greenwich, Conn.) thatproduces a chromophore following hydrolysis by thrombin. The rates ofhydrolysis of S238 by bovine thrombin alone (250 pM; FIG. 4, −saliva)and in the presence of horn fly saliva (equivalent to 2 flies) weremeasured at increasing concentrations of substrate over the range of2.5-100 μM. These data confirmed the observation that horn fly salivacontains an inhibitor of thrombin and indicate that it may be acompetitive inhibitor, as its effect is diminished when substrate isunlimited (100 μM). Several models can account for such biochemicalbehavior (Segel (1976) Biochemical Calculations, John Wiley & Sons, NewYork). For analysis, a Dixon plot is generated by determining thevelocity (v) of substrate hydrolysis by thrombin in the presence ofdifferent fixed concentrations of substrate, and plotting 1/v versusinhibitor concentration. This provides the means to identify the type ofinhibition and to determine the inhibition constant, Ki.

[0112] Characterization of the Physical Properties of the Anti-clottingComponent(s) in Horn Fly Salivary Glands to Devise a Purification Plan

[0113] APTT clotting times in Table 2 indicate that activity in SGE isdiminished after sitting at room temperature for 60 minutes or whensubjected to 100EC for 5 minutes. The activity precipitates withethanol, and is reasonably stable to treatment with acetonitrile/TFA andlyophilization. These physical attributes are consistent with aproteinaceous inhibitor that can be purified under standard HPLCprocedures using acetronitrile/TFA gradient elution. TABLE 2Characterization of the physical properties of anti-clotting activity inHaematobia irritans salivary gland extracts. Treatment APTT ClottingTime (Seconds) Control 52.3 SGE-Time O 62.6 SGE-room temperature × 60min 56.8 SGE-100° C. × 5 min 55.2 SGE-ethanol precipitate 59.8SGE-ethanol supernatant 50.1 SGE-lyopholized 57.0 SGE-50%acetonitrile/0.1% TFA 57.8

[0114] HPLC Purification and Recalcification Assay of HPLC SalivaFractions

[0115] For analytical method development, saliva from 100 to 150 flieswas pooled for each HPLC run. For preparative separation, saliva frommore than 500 flies was used for each run. Before injection onto thecolumn, pooled saliva was always diluted with the initial solvent of thepaired gradient A solvents. A macrosphere, C18, 4.6×250 mm, 300 Å column(AllTech) was used for all HPLC preparations. Protein elution wasmonitored by UV absorption at 220 nM, which detects peptide bonds.Components eluted from the column were collected at 0.5 or 1 minuteintervals. An aliquot for activity assays was transferred from eachfraction to a second tube containing bovine serum albumin (BSA) beforelyophilization of all samples to remove organic solvents. Fractionsdried with BSA (used to increase solubilization of purified protein)were reconstituted with Tris buffer (5 mM tris, 150 mM NaCl, pH 7.4 at37° C.). Inhibitory activity in fractions was defined by the delay inclot formation using the above-described recalcification assay.

[0116]FIG. 5 depicts the three reversed phase HPLC column proceduresused to obtain a highly pure preparation of anti-clotting activity.Black lines are HPLC chromatograms, while the gray bars indicateclotting times of recalcification assay. Panel A shows HPLC separationof H. irritans saliva using gradient elution (acetonitrile, 2-propanol,and TFA). Panel B shows HPLC separation of fraction with maximumanticlotting activity in “A” using gradient elution (acetonitrile andTFA). Panel C shows HPLC separation of fraction with maximumanticlotting activity in “B” using gradient elution (acetonitrile andHC1). Clotting data from the first fractionation run (A) indicated thathorn fly saliva contains only one clotting inhibitor that elutes atapproximately 45 minutes under the conditions used. For secondary HPLCseparation, fractions from the target peak were combined and injecteddirectly onto the column after the column had been equilibrated with theinitial solvent. Anti-coagulant activity was retained after 3consecutive HPLC runs, vacuum drying, and storage for 4 days at 4EC.

[0117]FIG. 6 shows SDS-PAGE of horn fly salivary anticlotting proteinafter the 3-step HPLC separation. Lane 1 contained protein concentrationmarker; lane 2, protein molecular weight standard marker; lane 3, theHPLC fraction with higher anticlotting activity (FIG. 5-C) and lane 4,the HPLC fraction with lower anticlotting activity (FIG. 5-C). Thisprofile indicated a single protein of high purity with a relativemobility of ≈16.5 KDa.

[0118] Construction of a Horn Fly Salivary Gland cDNA Library

[0119] Total salivary gland RNA (stored in several aliquots at −70EC fora period of ≈3 years) was thawed, pooled and mRNA isolated using poly(A)Quick^(R) reagents (Stratagene, LaJolla, Calif.). A cDNA library wasconstructed using a ZAP express™ vector and kit from Stratagene.Preliminary analysis of numbers of inserts indicated that a relativelysmall number of primary inserts was obtained (≈3×10⁴). Approximately ⅕of the primary library was reserved and the remainder used for one roundof amplification to yield a titer of 5.1×10⁶ plaque forming units (PFU)per ml.

[0120] Cloning the cDNA Coding for Thrombostasin

[0121] An estimated 110 pmoles of HPLC-pure thrombostasin were sent toHarvard Microchemistry Lab to obtain a precise molecular mass by massspectroscopy and identification of 30 residues of the amino-terminal(N-) sequence. Although our analysis by SDS/PAGE consistently indicateda mass of ≈16.5 KDa (see, for example, FIG. 6), mass spectroscopy of theHPLC-pure sample detected an apparent “family” of 4 proteins with anaverage mass of 9.3±0.06 KDa. One N-terminal sequence was obtained fromthe ˜9 KDa protein (SEQ ID NO: 3), indicating that the variable masseswere obtained from largely identical proteins that may have variable ionpairs or that differ by as few as 1-2 amino acids. The sequence from theN-terminus also suggested that the protein is highly acidic. A secondsample of thrombostasin, which was purified by HPLC and sent foranalysis, yielded a similar mass. The unused remainder of this secondsample was re-analyzed by SDS/PAGE. Again, the protein ran as a ˜16.5mass. Search of the scientific literature revealed another report ofhighly acidic protein that produced an anomalously high molecular masswhen analyzed by PAGE (Takano et al. (1988) Biochemistry 27:1964-1972).In order to confirm the molecular mass, a third batch of thrombostasinwith confirmed activity in a re-calcification assay, was subjected toSDS/PAGE. The single band of ˜16.5 KDa protein was transferred to a PVDFmembrane. The blot was stained with ponceau S to reveal the transferredthrombostasin band. This band and a control region of similar area wasexcised and sent to the Harvard Lab for sequence analysis. TheN-terminal sequence from this analysis (SAGPI) confirmed the identity ofthe first 5 amino acids of the N-terminus.

[0122] The N-terminal sequence obtained from the first 30 residues ofthrombostasin as set forth in SEQ ID NO: 3 was used to constructdegenerate nucleotide primers by the Scott-Ritchey Research Center(SRRC) DNA lab at Auburn University. For template DNA, an aliquot of theHaematobia irritans salivary gland cDNA was used that had been removedand frozen at −20EC following first strand cDNA synthesis for theabove-described library construction. A PCR reaction using thistemplate, the degenerate forward primer designed from thrombostasinN-terminal sequence and a reverse primer of oligo dT, yielded a productof approximately 350 base pairs. A 1 μl aliquot of the PCR product wasused in a ligation reaction with the PCR 2.1 vector (InvitrogenCorporation, San Diego, Calif.) at 14EC overnight. OneShot™ cells(Invitrogen Corporation) were then transformed with the ligation productand transferred onto LB agar plates containing ampicillin. Followingovernight growth, blue and white colonies were visible representingcells containing plasmid without an insert (blue) and plasmids with aninsert that disrupted the beta-galactosidase gene (white colonies). Tenwhite and 2 blue colonies were picked for amplification in liquidculture by overnight growth at ˜30EC. Aliquots of each culture werepreserved by storage in glycerol at −70EC. Plasmid size was estimatedvisually by ethidium bromide staining and comparison to molecular weightmarkers. DNA minipreps were prepared and sequenced by the SRRC DNA labusing primers based on sequences in the plasmid vector flanking themultiple cloning insertion site.

[0123] Analysis of the deduced amino acid sequence of the protein, setforth in SEQ ID NO: 2, coded for by the PCR-cloned cDNA set forth in SEQID NO: 1, confirmed identity to thrombostasin; i.e. the cDNA codes for a˜9 KDa protein and includes all the amino acids revealed by N-terminalsequencing, even though only a portion of that information was used inthe synthesis of degenerate primers that permitted amplification by PCR.Twenty-one percent of the putative protein is comprised of aspartic andglutamic acid residues. This information also confirmed that the cDNAencoding active thrombostasin is contained in the H. irritans cDNAlibrary. A search of protein databases in GenBank revealed no similarsequences.

[0124] Preparation of a Digoxigenin-Labeled Thrombostasin Probe

[0125] The above-described PCR-cloned thrombostasin cDNA fragment wasused to produce a digoxigenin-labeled probe for screening the H.irritans cDNA library under very stringent conditions. Adigoxigenin-labeled primer was synthesized by PCR using the clonedthrombostasin fragment as template and the Genius™ system (BoehringerMannheim, Indianapolis, Ind.) in a 1:5 digoxigenin-11-dUTP to dTTPratio. The digoxigenin-labeled DNA was purified by agarose gelelectrophoresis. Yield of labeled probe was estimated by titration andvisual comparison to a DIG-labeled control DNA provided in the GeniusKit.

[0126] Cloning and Sequencing of a Full-Length cDNA

[0127] XL1 blue cells were transfected with 50,000 plaque forming units(pfu) from the amplified library and plated on a 150-mm NZY plate.Following overnight incubation, the plate was chilled for 2 hr at +4ECbefore plaque lifts made in duplicate with nylon membranes and probedwith the digoxigenin-labeled DNA fragment. In brief, “lifted” DNA wasdenatured for 5 min at RT, dried for 5 min, neutralized 5 min and crosslinked in a Stratalinker 1800 (Stratagene, La Jolla, Calif.) on autolinkcycle; pre-hybridization and hybridization was in 5×SSC, 0.1%N-lauroylsarcosine, 0.02% SDS, 2% blocking reagent and 50% formamide at65EC; membranes were washed 4 times before visualization of thehybridized DIG-thrombostasin by incubation with anti-digoxigeninconjugated to alkaline phosphatase followed by substrate which producesa blue colored product. Several plaque picks from the first screeningwere subcloned to confirm positive clones in a secondary screen. Phagewas extracted from the plaque picks in SM buffer and amplified by growthin XL1-Blue MRF cells on NZY plates as described above. DNA was isolatedin minipreps of bacterial colonies grown overnight. Positive clones weretested by PCR amplification with thrombostasin-specific forward andreverse internal primers which were synthesized based on sequence in thecloned PCR fragment. Positive clones were further tested by anadditional plaque assay and shown to be pure by hybridization of allcolonies with the DIG/labeled probe.

[0128] Phagemids containing cloned inserts were obtained by automaticexcision using the ExAssist/XLOLR system and protocol of Stratagene.Colonies were grown on LB-kanamycin plates and glycerol stocks preparedfor storage at −70EC. Similar colonies were picked for amplification byovernight growth. DNA was extracted in minipreps and analyzed byautomated cycle sequencing (SRRC) in the forward direction using primersT3 and thrombostasin-F1 and in the reverse directions using primers T7and thrombostasin-R1, and with forward and reverse primers to sequencesinternal to the termini. Several cDNA clones were obtained andsequenced. The nucleotide sequences for a partial cDNA designated TB8are set forth in SEQ ID NO: 4, and the amino acid sequence encodedtherein are set forth in SEQ ID NO:5. It is noted that amino acidresidues 88-168 set forth in SEQ ID NO:5 encoded by nucleotides 263-505set forth in SEQ ID NO:4 correspond to active thrombostasin.

[0129] The Wisconsin Package™ of the Genetics Computer Group (GCG,Madison Wis.) was used to analyze nucleic acid and putative proteinsequences of thrombostasin cDNA.

[0130] To obtain the full length cDNA sequence, a 5′ RACE (RapidAmplification of cDNA Ends) procedure was employed, utilizing salivarygland mRNA and internal primers having 3′ consensus sequencecorresponding to the cDNA clones described above. Overlapping sequenceswere compiled to determine a composite full length cDNA sequence. ThecDNA clone TB8 described above was used to construct a full length cDNAencoding thrombostasin, by adapting the clone to contain the 5′ endnucleotides determined from the 5′ RACE procedure. The nucleotidesequences for the full length cDNA are set forth in SEQ ID NO:6 and theamino acid sequences encoded therein is set forth are SEQ ID NO:7. It isnoted that amino acid residues 95-175 set forth in SEQ ID NO: 7 encodedby nucleotides 283-525 set forth in SEQ ID NO:6 correspond to activethrombostasin.

[0131] Production of a Recombinant Thrombostasin (r-thrombostasin)Protein

[0132] Thrombostasin plasmid DNA and the transfer vector pBacPAK8(CLONTECH, Palo Alto, Calif.) were digested with 2 restriction enzymesthat cut in the plasmid's multi-cloning sites but not internal sequencesof thrombostasin. Excised thrombostasin and linearlized pBacPAK8 werepurified by TAE gel electrophoresis. Digested bands were excised and DNAextracted with “Sephaglas” Band Prep Kit (Pharmacia Biotech, Uppsala,Sweden). A 1:2 (vector:insert) ligation reaction was setup to runovernight at 15° C. OneShot™ cells were transformed with the BacPAK8plasmid containing the thrombostasin insert as described for the PCRfragment. Transformed cells were grown overnight on LB ampicillinplates. Several colonies were selected for liquid, overnight growth at37° C. Glycerol stocks were prepared and frozen at −70° C. and plasmidquick preps made for size evaluation by agarose gel visualization.Miniprep DNA was prepared by column purification (Qiagen Corp., SantaClarita, Calif.) for DNA sequencing using the Bac 2 primer (CLONTECH).

[0133] A recombinant baculovirus containing the thrombostasin insert wasgenerated by co-transfection of Sf9 cells with BacPAK8/thrombostasinplasmid TB8/3 and Bsu36I digested BacPAK6 viral DNA using lipofectin™(Life Technologies, Grand Island, N.Y.) as transfection reagent and HighFive™ Serum-Free Medium (Invitrogen, Carlsbad, Calif.). Controlsincluded wild type virus (positive control) and plasmid DNA only(negative control). Cells were incubated with transfection medium for 5hr at room temperature before adding TNM-FH medium containing 10% fetalbovine serum (TNM-FH/FBS), and further incubated at 27° C. for 72 hrs.Cell culture supernatant containing virus was collected and stored at 4°C. A plaque assay was performed to isolate pure thrombostasin-virusclones from the cell supernatant. Thirty-five mm plates containing1.5×10⁶ Sf9 cells each were infected in duplicate with 100 μl ofsupernatant or a dilution up to 10⁻³ in a 100 μl volume of TNM-FH/FBSmedium. After sitting for 1 hr at room temperature, infection medium wasremoved and the cells overlaid with 3.5 ml each of Grace's medium (LifeTechnologies, Grand Island, N.Y.), containing 10% FBS, 50 μg/mlGentamicin and 1% agarose. Cells were incubated in a plastic storage boxwith moist paper towels at 27EC. After 5 days, a second overlay wasadded that also included 0.16 mg/ml neutral red dye and 250 μg/ml Xgal.After the agarose overlay formed a gel, the dishes were inverted andincubated for 48 hr at room temperature. Clear, positive plaques werepicked and virus eluted by incubation overnight in TNM-FH medium. Sf9cells were infected with eluted virus and incubated for 4 days at 27ECto generate passage 1 virus.

[0134] Cells were collected in phosphate buffered saline (10 mM, pH 7.4)and DNA extracted using the Stratagene DNA micro extraction kit andprotocol II in the instruction manual. Extracted DNA was used astemplate for PCR with a thrombostasin forward and reverse primer pairand the Bac1/Bac2 primer pair (CLONTECH). Amplification with both primerpairs assured that the correct transformation event occurred. Asecondary plaque assay was conducted to assure clone purity, and todetermine virus titer.

[0135] Characterization of r-thrombostasin Production

[0136] Sf9 cells were infected with virus (multiplicity of infection=2)and incubated at 27° C. until media are collected at 12, 24, 48, 72 and96 hr. Virus is concentrated and removed from media by centrifugation inCentriplus™ 100 concentrators (Amicon, Beverly, Mass.) at 3,000×g for 2hr at room temperature. Total protein in the <100 KDa fraction isestimated by the modified Lowry Assay (Sigma Chemical, St. Louis, Mo.).

[0137] Anti-clotting and/or antithrombin activity of r-thrombostasin istested using the chromogenic substrate S238 assay as described above.

[0138] Purification of r-thrombostasin by RP/HPLC

[0139] Large molecular weight components (≧10 KDa) in the virus-freecell culture supernatant are concentrated by centrifugation at 3,000×gfor 4 hr in Centriplus™ 10 microconcentrators. RP/HPLC using a C18macrosphere column and elution with an acetonitrile gradient is used forisolation of r-thrombostasin from other medium components as describedabove.

[0140] Immunogenic properties of thrombostasin in a laboratory animalmodel (rabbits) as the first step toward production of a nucleic acid(DNA) vaccine against horn fly blood-feeding.

[0141] Anti-hemostatic proteins in the saliva of blood-feeding insectsare not highly immunogenic (see Cupp and Cupp (1997) J. Med. Entomol.34:87-94). This experimental observation agrees with the intuitiveconcept that generation of an immune response, especially production ofneutralizing antibody, might prevent or decrease blood feeding andproduction of progeny and fitness. Thus, it is important to developmethods to elicit a robust immune response to such molecules. Moreover,effective immunization of cattle in the field also requires a practicalvaccine that needs a minimum of handling and storage. In the past fewyears, immunization with nucleic acids has been demonstrated to generatestrong immune responses to encoded proteins that can be directed tospecific immune compartments by the location and/or amount of nucleicacid administered. Such vaccines, composed of plasmids with the DNA ofinterest inserted, can be produced at low cost and by relative simpletechniques of bacterial culture; they are stable to storage at roomtemperature and thus circumvent many of the problems of protein-basedvaccines. Thus, initially, immunization of rabbits is tested withthrombostasin nucleic acid.

[0142] A vaccine plasmid is constructed containing the CMV promoter andkanamycin resistance for selection. The procedures for restrictiondigestion and re-ligation of the baculovirus transfer vector asdescribed above is used to produce the thrombostasin containing vaccineplasmid (TS-Vac).

[0143] Serum, serving as pre-immunization control, is obtained fromblood samples taken from each rabbit via the large ear vein. Ninerabbits are injected intradermally with one of three TS-Vac plasmids inPBS (500 μg of plasmid with no insert=control; 200 μg, or 500 μgTS-Vac=test plasmids). Blood is sampled after approximately 4 weeks andtested for humoral (the presence of specific antibody titer) andcellular response (blastogenic response) to thrombostasin. Theseparameters are monitored on a weekly basis thereafter up to 20 weekspost-injection. All immunological assays used are standard and have beenused in previously published work on immune response to salivary factorsof blood-feeding insects (Cross et al. (1993) J. Med. Entomol.30:725-734; Cross et al. (1993) J. Med. Entomol. 30:928-935).

[0144] Antibody titer is determined by a direct ELISA assay (Cross etal. (1993) J. Med. Entomol. 30:725-734; Cross et al. (1993) J. Med.Entomol. 30:928-935). Briefly, 96 well flat-bottomed microtiter platesare coated with r-thrombostasin, or non-specific protein, then blockedwith 1% bovine serum albumin (BSA) in PBS. Wells are incubated withpre-immune sera or test sera, then washed with PBS-tween before additionof alkaline phosphatase-conjugated anti-rabbit IgG (SigmaImmunochemicals, St. Louis, Mo.) or anti-rabbit IgM (SouthernBiotechnology Assoc. Inc., Birmingham, Ala.) Following substratereaction with p-nitrophenyl phosphate (pNPP), color intensity is read at405 nm using a Spectramax microtiter plate reader (Molecular Devices,Sunnyvale, Calif.). Antibody titer is calculated and compared amongtreatments to determine the optimum amount of immunogen for antibodygeneration.

[0145] Antibody is evaluated for specificity to thrombostasin by dotblot (Cross et al. (1993) J. Med. Entomol. 30:725-734) usingr-thrombostasin. R-thrombostasin, or bovine serum albumin (BSA) controlis dotted onto wells of 96-well nitrocellulose-bottomed microtiterplates (Millipore, Marlborough, Mass.). Non-fat milk (3%) in PBS is usedas a blocking solution. Test sera are added and incubated for 1 hrbefore washing and substrate reaction. Specificity of reaction isdetermined by visual inspection.

[0146] Cellular response to thrombostasin is tested using peripheralblood mononuclear cells (PBM) isolated by Ficoll-Paque centrifugation ofblood collected in EDTA as anticoagulant (Ramachandra & Wikel (1992) J.Med. Entomol. 29:818-826). Viability of isolated cells is determined onan aliquot using fluorescein diacetate (FDA; Sigma, St. Louis, Mo.)which brightly labels healthy cells. PBM are added at 5×10⁵ cells/wellof a microtiter plate before addition of r-thrombostasin, horn flysaliva or non-specific protein. The mitogen ConA is added and incubationcontinued for 72 hr. Cellular response to test protein is determinedusing the MTT colorimetric assay (Denizot and Land (1986) J. Immunol.Methods. 89(2):271-277) which is read in the Spectormax at 570 nm.Blastogenic response to thrombostasin is determined by increase overcontrol. Comparison of response in the presence of whole saliva canmonitor for immunomodulating factors in saliva.

[0147] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0148] Other modifications and embodiments of the invention will come tomind in one skilled in the art to which this invention pertains havingthe benefit of the teachings presented herein. Therefore, it is to beunderstood that the invention is not to be limited to the specificembodiments disclosed. Although specific terms are employed, they areused in generic and descriptive sense only and not for purposes oflimitation, and that modifications and embodiments are intended to beincluded within the scope of the appended claims.

1 7 1 370 DNA Haematobia irritans CDS (1)...(243) 1 agt gcg ggt ccc atcaca ctg caa tta gat gat gat gat gat gac gac 48 Ser Ala Gly Pro Ile ThrLeu Gln Leu Asp Asp Asp Asp Asp Asp Asp 1 5 10 15 tct ggt atc ccc atattt gaa atg gat gat gaa gat gaa gac tct aat 96 Ser Gly Ile Pro Ile PheGlu Met Asp Asp Glu Asp Glu Asp Ser Asn 20 25 30 gac aat caa aaa ttt ccttta agt ttt gaa cgg ttt cca gaa aat gaa 144 Asp Asn Gln Lys Phe Pro LeuSer Phe Glu Arg Phe Pro Glu Asn Glu 35 40 45 aaa aat caa gta ggc ttg agagct aga ttt aac aaa ttc atg gca aaa 192 Lys Asn Gln Val Gly Leu Arg AlaArg Phe Asn Lys Phe Met Ala Lys 50 55 60 ttt act tcg ctg ttt ggc cgt cgtcgt ggc gta aat gtt ccc aat gct 240 Phe Thr Ser Leu Phe Gly Arg Arg ArgGly Val Asn Val Pro Asn Ala 65 70 75 80 gca taagcaaact aatattatatattaattact tcatttatgt gttctacact 293 Ala atataacaaa taaaaggattattaattaat tcataaaaaa aaaaaaaaaa aaaaaaaaaa 353 aaaaaaaaaa aaaaaaa 370 281 PRT Haematobia irritans 2 Ser Ala Gly Pro Ile Thr Leu Gln Leu Asp AspAsp Asp Asp Asp Asp 1 5 10 15 Ser Gly Ile Pro Ile Phe Glu Met Asp AspGlu Asp Glu Asp Ser Asn 20 25 30 Asp Asn Gln Lys Phe Pro Leu Ser Phe GluArg Phe Pro Glu Asn Glu 35 40 45 Lys Asn Gln Val Gly Leu Arg Ala Arg PheAsn Lys Phe Met Ala Lys 50 55 60 Phe Thr Ser Leu Phe Gly Arg Arg Arg GlyVal Asn Val Pro Asn Ala 65 70 75 80 Ala 3 30 PRT Haematobia irritans 3Ser Ala Gly Pro Ile Thr Leu Gln Leu Asp Asp Asp Asp Asp Asp Asp 1 5 1015 Ser Gly Ile Pro Ile Phe Glu Met Asp Asp Glu Asp Glu Glu 20 25 30 4611 DNA Haematobia Irritans CDS (2)...(505) 4 t gga atc tta gct ctt tcagct gtc tgc cag gcc caa aat gtc tta tca 49 Gly Ile Leu Ala Leu Ser AlaVal Cys Gln Ala Gln Asn Val Leu Ser 1 5 10 15 gga cgc cgc caa cat ggtgcc caa gga ctt tct gga tat tct ggt gat 97 Gly Arg Arg Gln His Gly AlaGln Gly Leu Ser Gly Tyr Ser Gly Asp 20 25 30 aat gac tgg gga tat tac ggtgaa gcc gga gct cca gga tcg gac tac 145 Asn Asp Trp Gly Tyr Tyr Gly GluAla Gly Ala Pro Gly Ser Asp Tyr 35 40 45 tct ggt tct tca ggt caa tgg gcaccc tta gat ttt gat tat aac agt 193 Ser Gly Ser Ser Gly Gln Trp Ala ProLeu Asp Phe Asp Tyr Asn Ser 50 55 60 cta cct gga tta tcg gga tat aac catgaa caa caa gat tac gaa gaa 241 Leu Pro Gly Leu Ser Gly Tyr Asn His GluGln Gln Asp Tyr Glu Glu 65 70 75 80 gat agt tat cgc cat gta cgc agt gcgggt ccc atc aca ctg caa tta 289 Asp Ser Tyr Arg His Val Arg Ser Ala GlyPro Ile Thr Leu Gln Leu 85 90 95 gat gat gat gat gat gac gac tct ggt atcccc ata ttt gaa atg gat 337 Asp Asp Asp Asp Asp Asp Asp Ser Gly Ile ProIle Phe Glu Met Asp 100 105 110 gat gaa gat gta gac tct aat gac aat caaaaa ttt cct tta agt ttt 385 Asp Glu Asp Val Asp Ser Asn Asp Asn Gln LysPhe Pro Leu Ser Phe 115 120 125 gaa cgg ttt cca gaa aat gaa aaa aat caagta ggc ttg aga gct aga 433 Glu Arg Phe Pro Glu Asn Glu Lys Asn Gln ValGly Leu Arg Ala Arg 130 135 140 ttt aac aaa ttc atg gca aaa ttt act tcgctg ttt ggc cgt cgt cgt 481 Phe Asn Lys Phe Met Ala Lys Phe Thr Ser LeuPhe Gly Arg Arg Arg 145 150 155 160 ggc gta aat gtt ccc aat gct gcataagcaaact aatattatat attaattact 535 Gly Val Asn Val Pro Asn Ala Ala 165tcatttatgt gttctacact atataacaaa taaaaggatt attaattaat tcataaaaaa 595aaaaaaaaaa aaaaaa 611 5 168 PRT Haematobia Irritans 5 Gly Ile Leu AlaLeu Ser Ala Val Cys Gln Ala Gln Asn Val Leu Ser 1 5 10 15 Gly Arg ArgGln His Gly Ala Gln Gly Leu Ser Gly Tyr Ser Gly Asp 20 25 30 Asn Asp TrpGly Tyr Tyr Gly Glu Ala Gly Ala Pro Gly Ser Asp Tyr 35 40 45 Ser Gly SerSer Gly Gln Trp Ala Pro Leu Asp Phe Asp Tyr Asn Ser 50 55 60 Leu Pro GlyLeu Ser Gly Tyr Asn His Glu Gln Gln Asp Tyr Glu Glu 65 70 75 80 Asp SerTyr Arg His Val Arg Ser Ala Gly Pro Ile Thr Leu Gln Leu 85 90 95 Asp AspAsp Asp Asp Asp Asp Ser Gly Ile Pro Ile Phe Glu Met Asp 100 105 110 AspGlu Asp Val Asp Ser Asn Asp Asn Gln Lys Phe Pro Leu Ser Phe 115 120 125Glu Arg Phe Pro Glu Asn Glu Lys Asn Gln Val Gly Leu Arg Ala Arg 130 135140 Phe Asn Lys Phe Met Ala Lys Phe Thr Ser Leu Phe Gly Arg Arg Arg 145150 155 160 Gly Val Asn Val Pro Asn Ala Ala 165 6 631 DNA HaematobiaIrritans CDS (1)...(525) 6 atg aag cat ttc gta gtt att gga atc tta gctctt tca gct gtc tgc 48 Met Lys His Phe Val Val Ile Gly Ile Leu Ala LeuSer Ala Val Cys 1 5 10 15 cag gcc caa aat gtc tta tca gga cgc cgc caacat ggt gcc caa gga 96 Gln Ala Gln Asn Val Leu Ser Gly Arg Arg Gln HisGly Ala Gln Gly 20 25 30 ctt tct gga tat tct ggt gat aat gac tgg gga tattac ggt gaa gcc 144 Leu Ser Gly Tyr Ser Gly Asp Asn Asp Trp Gly Tyr TyrGly Glu Ala 35 40 45 gga gct cca gga tcg gac tac tct ggt tct tca ggt caatgg gca ccc 192 Gly Ala Pro Gly Ser Asp Tyr Ser Gly Ser Ser Gly Gln TrpAla Pro 50 55 60 tta gat ttt gat tat aac agt cta cct gga tta tcg gga tataac cat 240 Leu Asp Phe Asp Tyr Asn Ser Leu Pro Gly Leu Ser Gly Tyr AsnHis 65 70 75 80 gaa caa caa gat tac gaa gaa gat agt tat cgc cat gta cgcagt gcg 288 Glu Gln Gln Asp Tyr Glu Glu Asp Ser Tyr Arg His Val Arg SerAla 85 90 95 ggt ccc atc aca ctg caa tta gat gat gat gat gat gac gac tctggt 336 Gly Pro Ile Thr Leu Gln Leu Asp Asp Asp Asp Asp Asp Asp Ser Gly100 105 110 atc ccc ata ttt gaa atg gat gat gaa gat gta gac tct aat gacaat 384 Ile Pro Ile Phe Glu Met Asp Asp Glu Asp Val Asp Ser Asn Asp Asn115 120 125 caa aaa ttt cct tta agt ttt gaa cgg ttt cca gaa aat gaa aaaaat 432 Gln Lys Phe Pro Leu Ser Phe Glu Arg Phe Pro Glu Asn Glu Lys Asn130 135 140 caa gta ggc ttg aga gct aga ttt aac aaa ttc atg gca aaa tttact 480 Gln Val Gly Leu Arg Ala Arg Phe Asn Lys Phe Met Ala Lys Phe Thr145 150 155 160 tcg ctg ttt ggc cgt cgt cgt ggc gta aat gtt ccc aat gctgca 525 Ser Leu Phe Gly Arg Arg Arg Gly Val Asn Val Pro Asn Ala Ala 165170 175 taagcaaact aatattatat attaattact tcatttatgt gttctacactatataacaaa 585 taaaaggatt attaattaat tcataaaaaa aaaaaaaaaa aaaaaa 631 7175 PRT Haematobia Irritans 7 Met Lys His Phe Val Val Ile Gly Ile LeuAla Leu Ser Ala Val Cys 1 5 10 15 Gln Ala Gln Asn Val Leu Ser Gly ArgArg Gln His Gly Ala Gln Gly 20 25 30 Leu Ser Gly Tyr Ser Gly Asp Asn AspTrp Gly Tyr Tyr Gly Glu Ala 35 40 45 Gly Ala Pro Gly Ser Asp Tyr Ser GlySer Ser Gly Gln Trp Ala Pro 50 55 60 Leu Asp Phe Asp Tyr Asn Ser Leu ProGly Leu Ser Gly Tyr Asn His 65 70 75 80 Glu Gln Gln Asp Tyr Glu Glu AspSer Tyr Arg His Val Arg Ser Ala 85 90 95 Gly Pro Ile Thr Leu Gln Leu AspAsp Asp Asp Asp Asp Asp Ser Gly 100 105 110 Ile Pro Ile Phe Glu Met AspAsp Glu Asp Val Asp Ser Asn Asp Asn 115 120 125 Gln Lys Phe Pro Leu SerPhe Glu Arg Phe Pro Glu Asn Glu Lys Asn 130 135 140 Gln Val Gly Leu ArgAla Arg Phe Asn Lys Phe Met Ala Lys Phe Thr 145 150 155 160 Ser Leu PheGly Arg Arg Arg Gly Val Asn Val Pro Asn Ala Ala 165 170 175

That which is claimed:
 1. A substantially purified protein havingantithrombin activity, wherein said protein is isolated from thesalivary glands of a species of the suborder Cyclorrhapha.
 2. Theprotein of claim 1, wherein said protein is isolated from the salivaryglands of a species of Haematobia.
 3. The protein of claim 2, whereinsaid species is Haemotobia irritans.
 4. A substantially purified proteinhaving the amino acid sequence selected from the group consisting of: a)an amino acid sequence of an antithrombin protein from a species of thesuborder Cyclorrhapha; b) an amino acid sequence that is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 2, SEQ IDNO: 5, or SEQ ID NO: 7; c) an amino acid sequence that is at least 90%identical to the amino acid sequence set forth in SEQ ID NO: 2, SEQ IDNO: 5, or SEQ ID NO: 7; and d) an amino acid sequence that is at least95% identical to the amino acid sequence set forth in SEQ ID NO: 2, SEQID NO: 5, or SEQ ID NO: 7; and e) an amino acid sequence set forth inSEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:
 7. 5. A fragment of the aminoacid sequence according to SEQ ID NO:2, SEQ ID NO: 5, or SEQ ID NO: 7;wherein said fragment comprises at least 15 contiguous amino acids ofsaid sequence.
 6. The protein of claim 4, wherein said protein isproduced by recombinant methods.
 7. The protein of claim 4, wherein saidprotein is capable of modulating the immune response.
 8. Apharmacological composition comprising a therapeutically effectiveamount of the protein of claim
 4. 9. A veterinary vaccine comprising atherapeutically effective amount of the protein of claim
 4. 10. A methodof treating hematophagy in cattle, said method comprising administeringto said cattle the vaccine of claim
 9. 11. A method of treatingthrombosis in a mammal, said method comprising administering to saidmammal a therapeutically effective amount of a protein havingantithrombin activity, wherein said protein has an amino acid selectedfrom the group set forth in claim
 4. 12. A method of claim 11, whereinsaid protein is produced by recombinant methods.